Infection by hepatitis C virus (HCV) is a compelling human medical problem. HCV is the causative agent for most cases of non-A and non-B hepatitis, with an estimated prevalence of 170 million cases (about 2-3%) globally. Four million individuals may be infected in the United States.
Upon first exposure to HCV, about 10% of infected individuals develop acute clinical hepatitis. In most instances the virus establishes a chronic infection that persists for decades. This usually results in recurrent and progressively worsening liver inflammation, which can lead to a more severe disease state such as cirrhosis and hepatocellular carcinoma.
HCV is an enveloped positive-stranded RNA virus belonging to the Flaviviridae family, and has a genome that encodes a polyprotein of 3010 to 3033 amino acids. The HCV nonstructural (NS) proteins provide the catalytic machinery for viral replication, and are derived by proteolytic cleavage of the polyprotein. A vital enzyme encoded by HCV is NS5B replicase protein, which has RNA-dependent RNA polymerase (RdRp) function and is believed to be responsible for HCV genome replication.
Currently, there are no broadly effective treatments for the debilitating progression of chronic HCV. An understanding of the differences between HCV replicase and cellular polymerases will play an essential role in elucidating the molecular basis of HCV polymerase action to facilitate the design of specific inhibitors against HCV replication. The unique structural features of HCV NS5B replicase [see, e.g., Bressanelli et al., Proc. Natl. Acad. Sci USA 96:13034-13039 (1999); Ago et al., Structure 7: 1417-1426 (1999); Lesburg et al., Nat. Struct. Biol. 6: 937-943 (1999)], in combination with detailed kinetic information about its mechanism of action will help to design specific inhibitors against this polymerase without also targeting cellular polymerases.
The conventional replicase assays used to detect HCV NS5B activity are based on incorporation of radioactive nucleotide substrate into a nascent RNA product. These assays use larger and less defined viral RNA or artificial homopolymeric RNA templates to measure the cumulative incorporation of nucleotides and the average steady state catalytic activity of the polymerase [Behrens et al., EMBO J. 15: 12-22 (1996); De Francesco et al., Methods Enzymol. 275: 58-67 (1996); Ferrari et al., J. Virol. 73: 1649-1654 (1999); Lohmann et al., J. Virol. 71: 8416-8428 (1997)]. Mechanistic data for HCV NS5B-catalyzed nucleotide incorporation is lacking due to the lack of suitable RNA template and primer pairs. Thus, the mechanism of a polymerization reaction or a single turnover event (single nucleotidyl transfer reaction) by HCV replicase is yet unknown, because the conventional template and primer pairs do not assemble efficiently with the replicase enzyme to permit efficient nucleotide incorporation and extension of end-labeled primers.
The conventional assays are also unable to reveal the proportion of enzyme and RNA substrate that is involved in productive binding to form replicase complexes competent for catalysis. The relatively low replicase activity exhibited by these in vitro assays implies that a very small portion of the HCV replicase and RNA template assemble complexes that are competent for catalysis. The conventional assay systems are clearly not suitable for defining the kinetic and thermodynamic constants of HCV NS5B-catalyzed nucleotide incorporation, nor can they be used for mechanistic characterization of inhibitors that target the HCV polymerase protein.
Thus, there is a need in the art for an improved, more accurate assay system for detecting HCV replicase activity. An RNA template-primer pair that allows HCV NS5B to efficiently catalyze nucleotide extension of a labeled primer in a template-dependent fashion will be beneficial. To address this need, the present invention provides an assay system that uses small and well-defined synthetic RNAs which allow efficient assembly of all catalytic components in the quaternary complex for HCV NS5B-directed RNA replication.